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目的:采用反相高效液相色谱法,观察大黄素在Caco-2细胞中的摄取特点。方法:将大黄素与Caco-2细胞共同孵育,收集细胞样品,液氮反复冻融。取细胞裂解液,加入甲醇提取,提取液采用HPLC进行分析。色谱分析柱为C18柱(250mm×4．6mm,5μm,Diamonsil),流动相组成为85%乙腈及15%水(含0．1%乙酸),流速1ml·min-1,进样量20μl,柱温25℃,3D模式采集数据。结果:检测Caco-2细胞中大黄素的工作曲线的回归方程为Y=0．278x+0．148(Y=0．9996,n=5),线性范围为0．037～4．8μmol·L-1,最低检测浓度为0．018μmol·L-1。当细胞中大黄素的浓度为0．05、2和8．5μg·ml-1时,回收率分别为(101．3±7．3)%、(96．7±3．0)%和(98．7±2．1)%(n=5);相应的日内标准偏差分别为0．25%、2．9%和1．4%;相应的日间标准偏差分别为2．3%、5．6%和6．3%。大黄素在Caco-2细胞中的摄取达峰时间为10分钟,峰浓度为108．56±11．57 nmol/L·mg·protein,10分钟后Caco-2细胞中大黄素的含量迅速下降。浓度处于2-50μM之间时,Caco-2细胞对大黄素的摄取量呈线性增加,浓度达50μM后,随着剂量的增加大黄素的摄取量变化不明显。结论:大黄素可被Caco-2细胞迅速摄取,随着剂量的增加,大黄素在Caco-2细胞中的摄取存在饱和现象。
Objective: To observe the uptake characteristics of emodin in Caco-2 cells by RP-HPLC. Methods: The emodin and Caco-2 cells were incubated together to collect cell samples. The liquid nitrogen was freeze-thawed repeatedly. Cell lysates were taken and extracted with methanol. The extracts were analyzed by HPLC. The chromatographic column was a C18 column (250 mm×4.6 mm, 5 μm, Diamonsil). The mobile phase consisted of 85% acetonitrile and 15% water (containing 0.1% acetic acid). The flow rate was 1 ml·min-1. The injection volume was 20 μl. Column temperature 25 °C, 3D mode data collection. RESULTS: The regression equation for the detection of emodin in Caco-2 cells was Y=0.278x+0.148 (Y=0.9996, n=5), and the linear range was 0.037 to 4.8 μmol·L. -1, the lowest detection concentration was 0.018 μmol·L-1. When the concentration of emodin in the cells was 0.05, 2 and 8.5 μg·ml-1, the recoveries were (101.3±7.3)%, (96.7±3.0)%, and ( 98.7±2.1)% (n=5); the corresponding intraday standard deviations were 0.25%, 2.9%, and 1.4%, respectively; the corresponding daytime standard deviations were 2.3%, respectively. 5.6% and 6.3%. The peak uptake time of emodin in Caco-2 cells was 10 minutes and the peak concentration was 108.56±11.57 nmol/L·mg·protein. After 10 minutes, the content of emodin in Caco-2 cells rapidly decreased. When the concentration was between 2-50μM, the emodin uptake of Caco-2 cells increased linearly. After the concentration reached 50μM, the emodin uptake did not change significantly with increasing dose. Conclusion: Emodin can be rapidly taken up by Caco-2 cells. With the increase of dose, the uptake of emodin in Caco-2 cells is saturated.